Mycobacterium tuberculosis is the etiological agent of tuberculosis (TB) and is responsible for more than 1.7 million deaths and 8 million new infections annually. A state of emergency was recently declared by the World Health Organization (WHO) in Africa where TB has been associated with nearly one-third of all acquired immunodeficiency syndrome (AIDS)-associated deaths. Currently, 2 billion people are predicted to be persistently infected by M. tuberculosis worldwide. The processes that govern long-term infection by M. tuberculosis are complex and multifactorial, and they remain poorly understood. A better understanding of the molecular mechanisms regulating the establishment, maintenance, and reactivation of persistence by M. tuberculosis is urgently needed if TB is to be eradicated. MprAB has been shown to play a role in the establishment and maintenance of persistent infection by M. tuberculosis. mprAB encodes a two-component signaling system that is used to detect, integrate, and coordinate specific survival programs in response to stressful stimuli. Genes regulated by MprAB include mprAB itself, stress-responsive sigma factors sigE and sigB, a putative serine protease pepD, and the DosRST regulon. We hypothesize that the presence of misfolded or altered protein substrates induced in or at the cell surface following exposure of M. tuberculosis to stress activates MprAB and initiates downstream survival programs that include induction of a serine protease to degrade or refold altered protein substrate(s) and genes comprising the DosRST regulon to initiate a general survival response program. In this proposal, two specific aims have been proposed. The first aim will determine the biochemical function of PepD and its importance in M. tuberculosis physiology and pathogenesis. This will be accomplished by purifying various rPepD derivatives and examining their enzymatic activity. In addition, pepD mutant strains will be constructed and utilized in various in vitro and in vivo assays to investigate the importance of this gene in M. tuberculosis physiology and virulence. Finally, the natural substrate targets of PepD will be identified and verified using proteomic approaches. The second aim will investigate the role of MprAB in regulation of the DosRST regulon. This will be accomplished by characterizing and comparing promoter regions from MprA and DosR co- regulated genes, and examining the regulation of these genes in various M. tuberculosis derivatives during growth in macrophages in vitro and in a murine model of latent TB. In addition, consequences of mprA and/or dosR gene deletion on M. tuberculosis physiology and virulence will be assessed using in vitro survival assays and in vitro and in vivo model systems of infection. The proposal outlined here is expected to enhance our overall understanding of the factors contributing to M. tuberculosis persistence, including the genetic and environmental factors that regulate this dynamic process.